1. Field of the Invention
The present invention relates generally to methods and compositions that modulate C-reactive protein. Such modulators are useful for inhibiting C-reactive protein induced vascular inflammation and other inflammatory diseases.
2. Description of Related Art
Inflammatory response plays an important role in the onset, development and evolution of atherosclerotic lesions. Elevated serum levels of C-reactive protein (CRP) are non-specific but sensitive markers of the acute inflammatory response. A number of epidemiological studies have shown that the acute-phase reactant C-reactive protein is an important risk factor for atherosclerosis and ischemic heart disease. Higher levels of C-reactive protein are also related to increased risk of coronary events in patients with stable and unstable angina (Liuzzo et al., 1994). The basic mechanisms of this association are not clear and C-reactive protein can merely be a marker of inflammation, with no specific role in the pathogenesis of atherosclerosis. However, although C-reactive protein is present in atherosclerotic lesions, no previous study has specifically assessed the possible effects of C-reactive protein on vascular cells.
High levels of C-reactive protein are frequently observed in patients with unstable angina and acute myocardial infarction (Liuzzo et al., 1994). Patients with unstable angina levels  greater than 3 xcexcg/mL were associated with increased risk of coronary events (death, myocardial infarct and urgent coronary revascularization) and the association is even stronger for patients with  greater than 10 xcexcg/mL (Liuzzo et al., 1994). This observation has suggested that C-reactive protein is a risk factor for atherosclerosis and ischemic heart disease. These studies have shown that even small increases in the levels of C-reactive protein are associated with higher risk of atherosclerosis and ischemic heart disease in apparently healthy subjects (Ridker et al., 1997; Koeing et al., 1999p; Ridker et al., 2000) and the increased risk is independent of lipid-related and non-lipid-related cardiovascular risk. In patients with stable angina, levels of C-reactive protein  greater than 3.6 xcexcg/mL were associated with a two-fold increase in the risk of coronary events (Haverkate, et al., 1997).
C-reactive protein is an acute phase reactant protein usually present in human serum with a concentration of  less than 1 xcexcg/mL. However, C-reactive protein levels can increase up to 100 or even 500 times during acute inflammation. This staggering response is mainly regulated by proinflammatory cytokines, in particular interleukin-6, and is largely unaffected by anti-inflammatory drugs and hormones (Kilpatrick et al., 1991). Indeed, in patients with unstable angina with high C-reactive protein levels at discharge, C-reactive protein remains elevated during the follow-up and is associated with high risk of new coronary events, in particular in patients in the upper tertile of C-reactive protein levels ( greater than 8.6 xcexcg/mL) (Biasucci et al., 1999). In a recent large prospective study, patients with unstable angina and C-reactive protein levels of  greater than 15 xcexcg/mL at discharge had a 3-fold higher risk of coronary events during a 90-day follow-up (Ferreiros et al., 1999). These results suggest that the proinflammatory effects of C-reactive protein may contribute to the adverse outcome associated with higher levels of this acute phase reactant protein.
Although there is now strong evidence that C-reactive protein is an independent risk factor for ischemic heart disease (Shah 2000; Ridker et al., 2000), the mechanisms underlying this association are not clear. Since inflammatory responses play an important role in the development and evolution of atherosclerosis and may contribute to its thrombotic complications, C-reactive protein may merely be a marker of inflammatory response. Alternatively, C-reactive protein may have a direct role in the pathogenesis of atherosclerosis (Shah 2000; Lagrand et al., 1999). Due to its ligand binding properties, C-reactive protein plays a part in the innate immunity (opsonization) and in the removal of membrane and nuclear material from necrotic cells. C-reactive protein can also bind to complement factor C1q and factor H and activate the classic pathway of complement activation. In addition, recent studies have shown that C-reactive protein can bind to receptor FCxcex3RI (with low affinity) and FCxcex3RII (with high affinity) on leukocytes (Bharadwaj et al., 1999). Interestingly, C-reactive protein is present in atherosclerotic plaques but not in the normal vessel wall (Reynolds et al., 1987) where often colocalize with the terminal complement complex (Torzewski et al., 1998). C-reactive protein can also induce tissue factor expression by human monocytes (Cermak et al., 1993).
This invention relates generally to methods of screening for modulators of C-reactive protein (CRP). In certain embodiments of the invention, a composition of the modulator may be useful for inhibiting the development of C-reactive protein induced inflammatory diseases, e.g., cardiovascular diseases. The present inventors envision that blocking or lowering C-reactive protein levels may have beneficial effects on the evolution of atherosclerosis and may reduce the risk of coronary events.
In a specific embodiment of the present invention, it is provided a method of screening for modulators of C-reactive protein comprising: obtaining a C-reactive protein; contacting the C-reactive protein with at least a first candidate substance; and assaying for an interaction between the C-reactive protein and the first candidate substance with an assay. The assay may be used to assay for C-reactive protein induction of the expression of an adhesion molecule, a receptor, a signaling molecule, a cytokine or an enzyme. Exemplary adhesion molecules include, but are not limited to, intracellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM), or E-selectin. A specific example of a cytokine that is contemplated in the present invention is a chemokine, e.g., monocyte chemoattractant protein-1 (MCP-1). It is known that chemokines are small cytokines that are involved in the migration and activation of cells, especially phagocytic cells and lymphocytes. Furthermore, the candidate substance may inhibit or enhance the C-reactive protein induced expression of the adhesion molecule. It can be appreciated that the modulator can modulate either C-reactive protein or a co-factor involved in the function of C-reactive protein. Further, co-factors may be isolated from serum.
In yet another embodiment, the assay endpoint comprises assaying for inducible nitric oxide synthase (iNOS) induction, receptor for advanced glycation endproducts, monocyte chemoattractant protein-1, P-selectin, endothelin-1, endothelin-receptor, interleukin-6 or heme oxygenase-1. One skilled in the art will recognize that a variety of assays may be used to assay these endpoints, such as, FACS, ELISA, Northern blotting and/or Western blotting.
In another specific embodiment, C-reactive protein can be obtained by expressing C-reactive protein in a transgenic cell or an animal; isolating the expressed C-reactive protein; procuring from serum (i.e., human serum); and procuring from cells. Further, the (a transgenic cell) cell comprises a recombinant nucleic acid sequence encoding a C-reactive protein, thus the C-reactive protein is expressed from the recombinant nucleic acid sequence.
A specific embodiment may include contacting the C-reactive protein with a first candidate substance by incubating a cell in a composition comprising C-reactive protein. One particular aspect includes that the C-reactive protein is expressed in the cell before contacting the C-reactive protein with a first candidate substance. Further, the cell is incubated with C-reactive protein and serum. The serum may be human serum. A skilled artisan will recognize that serums from other species may be utilized in the present invention, such as, bovine or guinea pig. The cell may be a human cell, such as a human umbilical vein endothelial cell. It is within the scope of the present invention that other cells may be used.
In a further embodiment, the cell may be comprised in an animal. The animal may be a mammal, such as, a human. Other exemplary mammals that can be used in the present invention, include, but are not limited to, mice, rats, dogs, cats, guinea pigs, rabbits and monkeys.
In another embodiment, the C-reactive protein or the first candidate substance may be injected into the animal. The first candidate substance may be comprised in serum, such as human or naturally occurring serum.
In yet another embodiment, the first candidate substance may be admixed with serum prior to contacting the C-reactive protein with the first candidate substance.
In a specific embodiment, the identity of the first candidate substance may be known prior to performance of the screening method. The first candidate substance may be comprised in a mixture of possible candidate substances.
In a further embodiment, the identity of the first candidate substance may be unknown prior to performance of the screening method. The identity and characteristics of the first candidate substance may be determined after the performance of the screening method. For example, the first candidate substance may be isolated after the performance of the screening method. Exemplary isolation procedures include, but are not limited to, gel-filtration chromatography, ion-exchange chromatography, immunoaffinity chromatography, hydrophobic chromatography, or aqueous-phase hydrophobic-interaction chromatography. Further, one skilled in the art would be able to utilize well-known methods to determine characteristics of a protein, i.e., electrophoresis, spectrophotometric analysis, or amino acid analysis. Furthermore, a skilled artisan will realize that the above procedures are not all inclusive, and one skilled in the art will be capable of modifying the above procedures or utilizing other well-known protein analysis procedures. Thus, it is well within the knowledge of a skilled artisan to optimize the procedures depending upon the nature of the protein, i.e., soluble protein, membrane associated protein or an insoluble protein.
In another embodiment of the present invention, also provided is a method of inhibiting C-reactive protein modulated inflammation comprising: obtaining a modulator of C-reactive protein identified by a method comprising: obtaining a C-reactive protein; contacting the C-reactive protein with at least a first candidate substance; assaying for an interaction between the C-reactive protein and the first candidate substance with an assay; incorporating the modulator of C-reactive protein in a pharmaceutically acceptable carrier to form a pharmaceutical composition; and administrating the pharmaceutical composition to a subject. The modulator may inhibit C-reactive protein induced inflammation. Further, the modulator may inhibit the development of cardiovascular complications. For example, the modulator may be given to subject with angina or myocardial infarction. Also, the modulator may be given to subject who is at risk of atherosclerosis or ischemic heart disease. In addition, the modulator inhibits the development of a stroke or other C-reactive protein induced inflammatory diseases e.g., rheumatoid arthritis, lupus and inflammatory bowel disease. The modulator may be given to the subject in a prophylactic manner. The modulator may be given in a single dose or a series of doses. Furthermore, the series of doses can be administered daily. One skilled in the art will realize that a either a variety of combinations can be utilized to administer the modulator to a patient. For example, a daily single dose may be administered or a series of doses may be administered several times throughout the day. The present invention is not construed to be limited to the specific times or doses that are specified. A skilled artisan will recognize that the times and doses may need to be altered depending upon the modulator and its characteristics or the pharmaceutical carrier characteristics that are best utilized for a given modulator.
In yet another embodiment, also provided is a modulator of C-reactive protein produced by a method comprising: obtaining a C-reactive protein; contacting the C-reactive protein with a candidate substance; assaying for an interaction between the C-reactive protein and the candidate substance; determining that the candidate substance is a modulator of C-reactive protein. The modulator may be comprised in a pharmaceutically acceptable carrier.
In still another embodiment, a C-reactive protein may be labeled prior to contacting the C-reactive protein with a first candidate substance. The labeled C-reactive protein may be utilized as a screening tool for a first candidate substance or a modulator.
In another embodiment, also provided is a method of screening for a modified modulator, wherein a first candidate substance is isolated comprising: obtaining a C-reactive protein; contacting the C-reactive protein with the first candidate substance; assaying for an interaction between the C-reactive protein and the first candidate substance to establish a baseline of a non-modified modulator; modifying the first candidate substance; contacting C-reactive protein with the modified first candidate substance; assaying for an interaction between the modified modulator in the presence of C-reactive protein and comparing the modified modulator interaction with the established baseline of the non-modified modulator. Modifying the first candidate substance comprises modification of the amino acid or nucleic acid sequence of the first candidate substance, such as, adhesion molecules, receptors, signaling molecules, cytokines, or enzymes. Exemplary modifications to the amino acid sequence include, but are not limited to, chemical mutagenesis, radiation mutagenesis, truncation of amino acids or point mutation of amino acids. In addition to chemical modifications, a skilled artisan will recognize that the modulator may be sensitive to temperature fluctuations, thus, the modulator may be modified using heat or cold. Further examples of the nucleic acid sequence of the first candidate substance comprises chemical mutagenesis, radiation mutagenesis, insertional mutagenesis, in vitro scanning mutagenesis or site-directed mutagenesis. One skilled in the art recognizes that variations of these standard, well-known modification procedures can be utilized in the present invention. Further, the modified nucleic acid sequence is inserted into an expression vector. The expression vector comprises a reporter molecule. The expression vector is transfected into cells, such as human umbilical vein endothelial cells. Further, the reporter molecule is measured for protein expression, protein activity or binding activity after transfection. One skilled in the art realizes that the reporter molecule that is used in the expression vector dictates the type of activity that is measured. Thus, the present invention can be modified to include any of the available reporter molecules known in the art. A skilled artisan recognizes that transient transfection and stable transfection can be used in the present invention. In a specific embodiment, the cells are embryonic stem cells, which after transfection are implanted into a blastocyst to produce a transgenic mouse. Furthermore, a skilled artisan is cognizant of the variety of methods to produce transgenic mice. Another example is that the modified nucleic acid sequence is injected into the embryo to produce a transgenic mouse. Thus, the present invention can be modified to develop a transgenic mouse by any of the available methods known in the art.
As used herein the specification, xe2x80x9caxe2x80x9d or xe2x80x9canxe2x80x9d may mean one or more. As used herein in the claim(s), when used in conjunction with the word xe2x80x9ccomprisingxe2x80x9d, the words xe2x80x9caxe2x80x9d or xe2x80x9canxe2x80x9d may mean one or more than one. As used herein xe2x80x9canotherxe2x80x9d may mean at least a second or more.
Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.